JP2010142215A - Plum lactic acid bacterium fermentation food or drink and method for producing the same - Google Patents
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Abstract
Description
本発明は、耐酸性を有し、ウメ果実の発酵に適するLactobacillus sp.に属する乳酸菌、ならびに、該乳酸菌を含有するウメの飲食品およびその製造方法に関する。 The present invention is a Lactobacillus sp. Which has acid resistance and is suitable for fermentation of ume fruit. Relates to a lactic acid bacterium belonging to the above, a food and drink of ume containing the lactic acid bacterium, and a method for producing the same.
ウメは有機酸(主にクエン酸、リンゴ酸)を多く含み、酸味が強く、加水し希釈を行ってもpHは3未満であり、乳酸発酵には適していない。ウメの乳酸発酵に関する技術として(特許文献1)、(特許文献2)があるが、いずれもpH調整を行っており、(特許文献1)では6.5、(特許文献2)では3.0〜5.5で、pH3未満の範囲では乳酸発酵できないことが示されている。またウメの発酵に適した乳酸菌株の選抜は検討されておらず、既存の乳酸菌では、目的とするウメの乳酸発酵飲食品を得るには未だ改良の余地が多く残されているのが現状である。 Ume contains a lot of organic acids (mainly citric acid and malic acid), has a strong acidity, has a pH of less than 3 even after being diluted with water, and is not suitable for lactic acid fermentation. Although there are (Patent Document 1) and (Patent Document 2) as technologies relating to lactic acid fermentation of ume, pH adjustment is performed for both, and (Patent Document 1) is 6.5, and (Patent Document 2) is 3.0. It is shown that lactic acid fermentation cannot be performed at a pH of less than 3 at ˜5.5. In addition, the selection of lactic acid strains suitable for ume fermentation has not been studied, and there is still much room for improvement in the existing lactic acid bacteria to obtain the desired ume lactic acid fermented food and drink. is there.
本発明が解決しようとする課題は、ウメ果実を原料に乳酸菌の持つ機能性を付与した飲食物を得ることを目的に、梅果実の発酵に適しする乳酸菌株を選定し、該乳酸菌株を用いた安価で日常的摂取が可能なウメ果実の飲食品を新たに提供することである。 The problem to be solved by the present invention is to select a lactic acid strain suitable for fermentation of plum fruit for the purpose of obtaining food and drink having the functionality of lactic acid bacteria using ume fruit as a raw material. It is to provide new foods and drinks of ume fruit that are inexpensive and can be consumed daily.
本発明は、上記した課題を解決するためになされたものであって、本発明者らは、目的とする乳酸菌をスクリーニングするに際し、次のような基準を新たに設定し、選定作業を行った。
すなわち、本発明者らは、食経験が長く安全性の高い乳酸菌として、伝統的な発酵食由来の乳酸菌のうち▲1▼胃酸耐性が高い、▲2▼低pHでの生育が良好である、▲3▼胆汁耐性が高い▲4▼ウメ果実の持つ機能性を損なわない、▲5▼ウメ果実もしくはウメ果汁での生育が良好で、発酵後の風味、嗜好性が優れる菌株の選定につき研究を重ねた結果、これらの条件に合致する菌株としてLactobacillus sp. FPL2(本菌株は、独立行政法人製品評価技術基盤機構特許微生物寄託センターNITE AP−692として寄託された。)を見出した。本菌株の菌学的性質は、以下のとおりである。The present invention has been made in order to solve the above-mentioned problems, and the present inventors newly set the following criteria and performed selection work when screening the target lactic acid bacteria. .
That is, the present inventors, as a lactic acid bacterium having a long dietary experience and high safety, among the lactic acid bacteria derived from traditional fermented foods, (1) high resistance to gastric acid, (2) good growth at low pH, (3) High bile resistance (4) Does not impair the functionality of ume fruit, (5) Researches on selection of strains that grow well in ume fruit or ume juice and have excellent flavor and taste after fermentation As a result of overlapping, Lactobacillus sp. We found FPL2 (this strain was deposited as NITE AP-692, a patent microorganisms deposit center NITE AP-692, an independent administrative agency). The mycological properties of this strain are as follows.
A.形態的性状
細胞形態:桿菌
運動性:なし
胞子の有無:なし
グラム染色性:陽性A. Morphological characteristics Cell morphology: Neisseria gonorrhoeae motility: None Spore presence: None Gram staining: Positive
B.生理学的性状(陽性:+、陰性:−、弱陽性:w)
カタラーゼ −
ガス産生 −
15℃での生育 +
45℃での生育 −
発酵形式 ホモ発酵
乳酸旋光性 DL
ペプチドグリカンタイプ DAP
好塩性・耐塩性 0〜7.5%での生育 +
10%での生育 −B. Physiological properties (positive: +, negative:-, weakly positive: w)
Catalase −
Gas production −
Growth at 15 ° C +
Growth at 45 ° C-
Fermentation type Homofermentation lactic acid rotatory DL
Peptidoglycan type DAP
Saltiness and salt tolerance Growth at 0-7.5% +
Growth at 10%-
C.炭水化物発酵性(陽性:+、陰性:−、弱陽性:w)
アラビノース −
リボース +
キシロース −
グルコン酸 +
グルコース +
フルクトース +
ガラクトース +
マンノース +
ラムノース −
セロビオース +
ラクトース +
マルトース +
メリビオース +
スクロース +
ラフィノース +
サリシン +
トレハロース +
メリチトース −
マンニトール +
ソルビトール +
スターチ −
イヌリン −
グリセロール −C. Carbohydrate fermentability (positive: +, negative:-, weakly positive: w)
Arabinose −
Ribose +
Xylose −
Gluconic acid +
Glucose +
Fructose +
Galactose +
Mannose +
Rhamnose −
Cellobiose +
Lactose +
Maltose +
Melibiose +
Sucrose +
Raffinose +
Salicin +
Trehalose +
Merichitose −
Mannitol +
Sorbitol +
Starch −
Inulin −
Glycerol −
D.遺伝学的特性
BigDye Terminator v3.1 Cycle Sequencing Kitを用いた16SrDNAの全塩基配列はLactobacillus pentosus および Lactobacillus plantarumの16SrDNAに対し、相同率99.6%以上の高い相同性を示した。D. Genetic Characteristics The entire base sequence of 16SrDNA using BigDye Terminator v3.1 Cycle Sequencing Kit has a high homology of at least 99.6% compared to 16SrDNA of Lactobacillus pentosus and Lactobacillus plantarum.
E.胃酸耐性
胃酸耐性試験は以下の通りに実施した。人工胃液(100mM HCl/KCl buffer pH2.0 with 0.04% Pepsin)4.5mlに乳酸菌培養液(GYPブロス 30℃ 24hr)0.5mlを加え、37℃で3時間放置し、初発菌数および人工胃液に接触後の菌数をMRS agarを用いて計測し、生残率を算出した。本法により、本菌株は高い生存率を示し、接種後の生菌数を経時的にみたところ、図1に示すようにほとんど低下しなかった。E. Gastric acid resistance The gastric acid resistance test was performed as follows. 0.5 ml of lactic acid bacteria culture solution (GYP broth 30 ° C. 24 hr) is added to 4.5 ml of artificial gastric fluid (100 mM HCl / KCl buffer pH 2.0 with 0.04% Pepsin) and left at 37 ° C. for 3 hours. The number of bacteria after contact with the artificial gastric juice was measured using MRS agar, and the survival rate was calculated. By this method, the present strain showed a high survival rate, and when the number of viable bacteria after inoculation was observed over time, it hardly decreased as shown in FIG.
F.胆汁耐性
胆汁耐性試験は以下の通りに実施した。GYPブロスにて24時間前培養を行った菌株を0,0.1,0.2,0.3,0.4%胆汁末含有GYPブロスに接種、30℃で5日間培養、培養液2mlを0.1M水酸化ナトリウム溶液で滴定し、生育を判定した。胆汁濃度が高くなると、菌の生育は悪くなるが、図2に示すように本菌株は胆汁濃度0.4%下でも生育を示した。F. Bile tolerance The bile tolerance test was performed as follows. Inoculate GYP broth containing 0,0.1,0.2,0.3,0.4% bile powder for 24 hours pre-culture in GYP broth, culture at 30 ° C. for 5 days, 2 ml of culture solution Titration with 0.1 M sodium hydroxide solution was performed to determine growth. As the bile concentration increased, the growth of the bacteria worsened, but as shown in FIG. 2, the strain showed growth even under a bile concentration of 0.4%.
G.ウメ果実の発酵性
ウメ果実の発酵性試験は以下の通りに実施した。まず、冷凍保存しておいたウメ果実(品種:紅サシ)を自然解凍後圧搾し、果汁を調製した。このウメ果汁を2倍に希釈、グルコースを2%になるよう添加、クエン酸ナトリウムでpHを3.0に調整したものと、pH未調整(pH2.7)のままのものを、0.45・mメンブレンフィルターで濾過滅菌した。この滅菌ウメ果汁液に、GYPブロスにて24時間前培養を行った菌株を1/1000容接種し、30℃で発酵させた。図3に示すように、本菌株はpH未調整(pH2.7)のウメ果汁中においても、ほぼ接種した菌数を維持し、pH3.0の果汁においては菌数の増加を示した。G. Fermentability of ume fruit The fermentability test of ume fruit was performed as follows. First, the ume fruit (variety: Red Sashi) that had been frozen and frozen was naturally thawed and then pressed to prepare fruit juice. This ume juice was diluted 2 times, glucose was added to 2%, pH adjusted to 3.0 with sodium citrate, and pH unadjusted (pH 2.7) 0.45 • Sterilized by filtration with m membrane filter. This sterile ume fruit juice was inoculated with 1/1000 volume of a strain pre-cultured for 24 hours in GYP broth and fermented at 30 ° C. As shown in FIG. 3, this strain maintained almost the number of inoculated bacteria even in the ume juice without pH adjustment (pH 2.7), and showed an increase in the number of bacteria in the fruit juice at pH 3.0.
H.抗酸化機能の維持
ウメ果実にはクロロゲン酸や(+)−カテキンなどのポリフェノールが含まれ、抗酸化機能を有することが知られているが、乳酸発酵でこの抗酸化能力が維持できるかを検討した。上記ウメ果汁発酵液を用い、Troloxを標準物質に、DPPHラジカル消去活性を(非特許文献1)に従って測定したところ、図4に示すように、本菌株で発酵したウメ果汁の抗酸化能力は高まることがわかった。
Lactobacillus sp. FPL2は、ウメ果実での生育が良好で、ウメ果実の持つ抗酸化機能を高め、胃酸耐性、胆汁耐性を有し摂取後の生残性も期待でき、該菌株の生菌またはウメ果実の発酵物を食品(液状、凝固、ペースト状、凍結、乾燥品)として提供することが可能な菌株として選択したものである。そこで、Lactobacillus sp. FPL2株を用いたウメ発酵飲食物の製造特性を例を挙げて詳細に説明するが、本発明はこれらに限定されるものではない。 Lactobacillus sp. FPL2 has good growth in ume fruit, enhances the antioxidant function of ume fruit, has gastric acid resistance and bile resistance, and can be expected to survive after ingestion. The product is selected as a strain that can be provided as food (liquid, coagulated, pasty, frozen, dried). Therefore, Lactobacillus sp. Although the manufacturing characteristic of the ume fermentation food / beverage using FPL2 strain | stump | stock is demonstrated in detail using an example, this invention is not limited to these.
すなわち、本発明は ウメ果実に乳酸菌が利用できる糖類、水を加え、ウメ果実発酵に適したLactobacillus sp. に属する乳酸菌、該乳酸菌含有物、その処理物の少なくともひとつを含有してなること、を特徴とする飲食品に関するものである。乳酸菌含有物としては、乳酸菌懸濁液、乳酸菌培養物、乳酸菌培養物から固形分を除去した乳酸菌培養液、ウメ果実の発酵飲料などが挙げられる。 That is, the present invention adds Lactobacillus sp. Suitable for ume fruit fermentation by adding sugar and water that can use lactic acid bacteria to ume fruit. It is related with the food-drinks characterized by including at least one of the lactic acid bacteria which belong to this, the lactic acid bacteria containing material, and its processed material. Examples of the lactic acid bacteria-containing material include lactic acid bacteria suspensions, lactic acid bacteria cultures, lactic acid bacteria culture solutions obtained by removing solids from lactic acid bacteria cultures, and ume fruit fermented beverages.
処理物としては、固形物、凝固物、濃縮物、ペースト化物、乾燥物、液状物、希釈物、凍結物等が挙げられる。乳酸菌としては生菌体、湿潤菌体、乾燥菌体などが適宣使用可能である。 Examples of processed products include solids, coagulated products, concentrates, pasted products, dried products, liquid products, diluted products, frozen products, and the like. As the lactic acid bacteria, live cells, wet cells, dry cells, etc. can be suitably used.
本発明によれば、ウメ果実の果汁に糖類を添加した飲料や、梅干しなどの梅漬けに乳酸菌を添加して発酵させることも可能で、ウメ果実の飲料や漬物等を乳酸発酵させることができる。 According to the present invention, it is possible to ferment by adding lactic acid bacteria to a beverage obtained by adding saccharides to the fruit juice of ume fruit or pickled ume such as umeboshi.
また、本菌株は、ウメ果実に含まれるリンゴ酸を資化することがわかっており、ウメ果実中のリンゴ酸を低下させ、果実の酸味を調節することもできる。 Moreover, this strain is known to assimilate malic acid contained in ume fruit, and can also reduce malic acid in ume fruit and adjust the acidity of the fruit.
そして、胃酸耐性、胆汁耐性を有し摂取後の生残性も期待できる乳酸菌を用いることで保健機能を併せ持つウメの健康志向飲食品の製造が可能となる。 By using a lactic acid bacterium that has gastric acid resistance and bile resistance and can be expected to survive after ingestion, it is possible to produce a ume health-oriented food and drink having both health functions.
原料のウメはpHを調整すれば、品種や熟度に関係なく使用できるが、ウメ果実に含まれている有機酸含量は、品種や熟度に左右され、福井県で栽培されている品種「紅サシ」は、他のウメ品種に比べ、有機酸含量はやや低く、さらに完熟黄化することにより低くなるため、完熟黄化した「紅サシ」を用いれば、希釈のみでpHを調整しなくても乳酸発酵を行うことが出来る。pH調整を行い、pHを3近くまで上げると、再現よく乳酸発酵することが出来る。 If the pH of the raw ume is adjusted, it can be used regardless of the variety and maturity, but the organic acid content contained in the ume fruit depends on the variety and maturity, and the variety cultivated in Fukui Prefecture “ “Red Sashi” has a slightly lower organic acid content than other ume varieties and lowers when it is fully ripe yellowed. Even lactic acid fermentation can be performed. By adjusting the pH and raising the pH to nearly 3, lactic acid fermentation can be performed with good reproducibility.
飲食物製造のための乳酸菌株の前培養は、遠心分離などで培養菌体だけを集菌し、培地成分を除去して使用することのほか、食品として利用できる食品添加物としての酵母エキス、グルコースのみからなる液体培地でも十分生育させることができる。また、ウメの乳酸発酵物をそのままスターターとして添加することも可能である。添加量は原料の1/10000から1/10の範囲で使用することができる。 Pre-culture of lactic acid strains for food and beverage production involves collecting only cultured cells by centrifugation, etc., removing and using the medium components, yeast extract as a food additive that can be used as food, A liquid medium consisting only of glucose can be sufficiently grown. Moreover, it is also possible to add the lactic acid fermented product of a plum as a starter as it is. The addition amount can be used in the range of 1 / 10,000 to 1/10 of the raw material.
本乳酸菌の生育温度は15℃〜40℃、至適温度30℃で、この温度条件下で乳酸発酵を行うことができる。培養時間は30℃で2〜5日間で、発酵終了後は冷蔵(5℃)保存により、約1ヶ月後も乳酸菌数を維持することができる。乳酸発酵中に酵母汚染が懸念されるため、乳酸菌の添加前に原材料の加熱殺菌を行うことが望ましい。乳酸生菌を製品中に維持するには、発酵終了後は冷蔵、凍結、凍結乾燥などが有効であるが、死菌としてもよい場合には、乳酸発酵終了後に加熱殺菌を行ってもよい。 The growth temperature of the present lactic acid bacteria is 15 ° C. to 40 ° C., and the optimum temperature is 30 ° C., and lactic acid fermentation can be performed under this temperature condition. The culture time is 2 to 5 days at 30 ° C. After the fermentation, the number of lactic acid bacteria can be maintained even after about 1 month by refrigeration (5 ° C.) storage. Since there is concern about yeast contamination during lactic acid fermentation, it is desirable to heat sterilize the raw material before adding lactic acid bacteria. In order to maintain the lactic acid live bacteria in the product, refrigeration, freezing, lyophilization and the like are effective after completion of fermentation. However, when sterilization is acceptable, heat sterilization may be performed after completion of lactic acid fermentation.
飲料として提供する場合は、糖濃度、加水量を調節することで、製品の味を調節できる。乳酸菌添加時に寒天等のゲル化剤を添加することで、ヨーグルト状の凝固とて提供することもできる。また凍結させることでフローズンタイプのデザートとして提供することも可能であり、凍結乾燥などにより、粉末化し、サプリメントなどに利用することも可能である。 When provided as a beverage, the taste of the product can be adjusted by adjusting the sugar concentration and the amount of water added. By adding a gelling agent such as agar at the time of addition of lactic acid bacteria, it can also be provided as a yogurt-like coagulation. Moreover, it can be provided as a frozen type dessert by freezing, and it can be pulverized by lyophilization and used for supplements.
冷凍保存しておいたウメ果実(品種:紅サシ)を自然解凍後圧搾し、果汁を調製した。ウメ果汁を3倍に希釈し、グルコースを7%添加し、1%炭酸水素ナトリウムを0〜3ml加えpH2.85〜3.13まで変化させ、65℃、15分間加熱殺菌を行ない、GYPブロスにて24時間前培養を行ったFPL2株を1/500容接種し、吸光度(630nm)を測定し、濁度により生育を判定した。図5に示すように、pH低下に伴い、乳酸菌の生育は抑制されたが、pH3未満でも、吸光度の上昇が認められ、乳酸発酵を行うことが確認できた。 The ume fruit (variety: red sashi) that had been frozen and stored was naturally thawed and then pressed to prepare fruit juice. Dilute ume juice three times, add 7% glucose, add 0-3 ml of 1% sodium bicarbonate, change to pH 2.85-3.13, heat sterilize at 65 ° C. for 15 minutes, and add to GYP broth Then, 1/500 volume of FPL2 strain that had been pre-cultured for 24 hours was inoculated, the absorbance (630 nm) was measured, and the growth was determined by turbidity. As shown in FIG. 5, the growth of lactic acid bacteria was suppressed as the pH decreased. However, even when the pH was less than 3, an increase in absorbance was observed, confirming that lactic acid fermentation was performed.
試験例1に記載のウメ果汁に水、グルコース(終濃度1%) を加え、3倍希釈し、50mlずつわけ、65℃、15分間加熱殺菌を行ない、冷却後、FPL2株を1/500量加えて30℃のインキュベーターで発酵させ、乳酸菌数、有機酸含量を測定した。乳酸菌数はGYP白亜寒天培地を用い、有機酸は、下記に記載の島津有機酸分析システム(HPLC)により測定した。
《分離条件》
カラム:Shin−pack SCR−102H(8mmI.D.×300mmL.)
2本直列接続、カードカラム SCR−102H(6mmI.D.×50mmL.)付き
移動相:5mM p−トルエンスルホン酸水溶液
流量:0.8mL/min
温度:40℃
《検出条件》
試薬:5mM p−トルエンスルホン酸 および
100μ「M EDTA を含む
20mM Bis−tris水溶液
流量:0.8ml/min
検出器:CDD−6A
polarity :+
response :SLOW
temperature :43℃
scale :1×24μS/cm
図6に示すように乳酸菌を接種した直後のスタート時から3日目まではほとんど菌数変化が見られず106CFU/mL程であったが、3〜4日目にかけて菌数が増加し、5日で108CFU/mlに達した。その間の有機酸含量の変化を図7に示す。リンゴ酸は発酵開始直後から低下し4日目には消失した。クエン酸はわずかに低下傾向が認められ、乳酸は4日目までは徐々に増加しそれ以降大きく増加した。このウメ果汁の官能評価をおこなったところ、3日目では酸味がマイルドに感じられ、4日目以降は酸味が徐々に強まる傾向が認められ、有機酸含量の変化を反映していた。発酵時間を調節することで、ウメの酸味を調節できることが示唆された。Water and glucose (
<Separation conditions>
Column: Shin-pack SCR-102H (8 mm ID × 300 mm L.)
Two in series connection, with card column SCR-102H (6 mm ID × 50 mm L.) mobile phase: 5 mM p-toluenesulfonic acid aqueous solution flow rate: 0.8 mL / min
Temperature: 40 ° C
<Detection conditions>
Reagent: 20 mM Bis-tris aqueous solution containing 5 mM p-toluenesulfonic acid and 100 μM EDTA Flow rate: 0.8 ml / min
Detector: CDD-6A
polarity: +
response: SLOW
temperature: 43 ° C
scale: 1 × 24 μS / cm
As shown in FIG. 6, the number of bacteria was hardly changed from the start immediately after inoculation with lactic acid bacteria until the third day, and it was about 10 6 CFU / mL. However, the number of bacteria increased from 3 to 4 days. It reached 10 8 CFU / ml in 5 days. Changes in the organic acid content during that time are shown in FIG. Malic acid decreased immediately after the start of fermentation and disappeared on the fourth day. Citric acid showed a slight downward trend, and lactic acid gradually increased until
市販濃縮ウメ果汁(JA三方五湖 品種紅サシ 5倍濃縮 pH2.32)を200g、上白糖300g、水2,500gを加え、溶解後重曹(炭酸水素ナトリウム)4gを添加しpHを2.95に調整し、温浴上で70℃で20分間加熱殺菌し、室温に冷却後、本菌株の前培養液(GYPブロス30℃24時間培養)を3mL(1/1000容)添加し、30℃で2日間乳酸発酵してウメ乳酸発酵液状物を得た。 200 g of commercially available concentrated ume fruit juice (JA Mikata Goko varietal red sashi, 5-fold concentrated pH 2.32), 300 g of white sucrose, and 2,500 g of water were added. And then sterilized by heating at 70 ° C. for 20 minutes on a warm bath, cooled to room temperature, added with 3 mL (1/1000 volume) of the pre-culture solution (GYP broth 30 ° C. for 24 hours) at 30 ° C. A ume lactic acid fermentation liquid was obtained by lactic acid fermentation for 2 days.
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JP2010252726A (en) * | 2009-04-27 | 2010-11-11 | Kyushu Univ | Method for reinforcing anti-oxidizing activity function of persimmon and health food material |
KR101240725B1 (en) * | 2010-11-19 | 2013-03-07 | 웅진식품주식회사 | Method for preparing fermented solution of japanese apricot and japanese apricot beverage composition using fermented solution of japanese apricot prepared the method |
CN103876216A (en) * | 2012-12-21 | 2014-06-25 | 贵州天刺力食品科技有限责任公司 | Roxburgh rose fruit juice beverage and production method thereof |
KR20180057916A (en) * | 2016-11-23 | 2018-05-31 | 농업회사법인 주식회사 자연향기 | Manufacturing method of japanese apricot paste and manufacturing method of pudding, bread, cake and cookie using the same |
KR20180109369A (en) * | 2017-03-28 | 2018-10-08 | 농업회사법인 주식회사 자연향기 | Manufacturing method of seasoning granule using japanese apricot |
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JP2010252726A (en) * | 2009-04-27 | 2010-11-11 | Kyushu Univ | Method for reinforcing anti-oxidizing activity function of persimmon and health food material |
KR101240725B1 (en) * | 2010-11-19 | 2013-03-07 | 웅진식품주식회사 | Method for preparing fermented solution of japanese apricot and japanese apricot beverage composition using fermented solution of japanese apricot prepared the method |
CN103876216A (en) * | 2012-12-21 | 2014-06-25 | 贵州天刺力食品科技有限责任公司 | Roxburgh rose fruit juice beverage and production method thereof |
KR20180057916A (en) * | 2016-11-23 | 2018-05-31 | 농업회사법인 주식회사 자연향기 | Manufacturing method of japanese apricot paste and manufacturing method of pudding, bread, cake and cookie using the same |
KR20180109369A (en) * | 2017-03-28 | 2018-10-08 | 농업회사법인 주식회사 자연향기 | Manufacturing method of seasoning granule using japanese apricot |
KR101965150B1 (en) * | 2017-03-28 | 2019-04-05 | 농업회사법인 주식회사 자연향기 | Manufacturing method of seasoning granule using japanese apricot |
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